Human hemoglobins are tetramers of globin chains folded around four heme groups. The tetramer has a molecular weight of approximately 64,500. A functional hemoglobin (Hb) is composed of two alpha (α) globin chains and two non-alpha (β, γ or δ) chains. A total of eight functional globin chains are found in various stages of development, producing eight types of normal Hb tetramers. Adults have predominantly HbA (“normal” or “common” hemoglobin) and a small amount of HbA2. The hemoglobin of newborns is comprised mainly of fetal hemoglobin (HbF), of about ˜15-40% is HbA. Fetal hemoglobin comprises only a barely detectable level of HbA2.
As a result of mutations in the genes encoding the different Hb-chains, there are more than 700 known variant hemoglobins. The majority of these variants are due to substitutions of amino acids on a single globin chain. Most of the mutations produce no clinically significant abnormal Hb function. Less than ten of the variants cause severe disease conditions, so called hemoglobinopathies.
The variant hemoglobins are geographically unevenly distributed and occur at different frequency in different areas. The earliest identified and clinically most significant variant is HbS, which relates to sickle cell anemia. The HbS variant hemoglobin is traditionally most abundant in populations of African and Mediterranean ethnicity. Due to increased ethnic diversity in most countries hemoglobinopathies seem to become more common also in Europe and the U.S.A. The birth prevalence of sickle cell anemia has increased by almost 50% over one decade in U.K. and the overall estimated prevalence for U.K. (1:2380) is even higher than many other inherited diseases, such as cystic fibrosis or phenylketonuria. Sickle cell anemia is a very severe disease, approximately 20% of children with the disease die within the first two years, often by infections. An early neonatal identification of this hemoglobinopathy substantially decreases the mortality and morbidity during the first five years of life. Heterozygotes (HbAS) show no symptoms.
Other important Hb variants include HbE, which is the second most frequent hemoglobinopathy, and occurs predominantly in Asia. HbC occurs most frequently in western parts of Africa. Other common hemoglobin variants are HbG and HbD.
Some of the variant hemoglobins, such as HbS, occur at such a high frequency that routine screening of newborns to identify possibly afflicted subjects is recommended. In some areas, such as most states of the United States and in Brazil, all newborn babies are subjected to neonatal testing for possible sickle trait, and other countries are considering adding universal neonatal screening programs to their national health care programs. Universal neonatal hemoglobinopathy screening programs are recommended in e.g., United Kingdom in areas where the minority ethnic population of African origin exceeds 15%. Central Middlesex Hospital in northwestern London tests all babies born in the North Thames (West) healt region, i.e., ˜50 000 births per year (Campbell et al. in Clin Chem, 45:7, 969-975, 1999).
Other hemoglobin variants considered to be included in routine neonatal screening are e.g., HbC, HbD-Punjab, and Hb-Barts. All of these hemoglobin variants are characterized by mutations leading to amino acid substitutions in the β-chain. Abnormal hemoglobins due to mutations in the α-chain are relatively uncommon.
Thalassemias are diseases caused by mutations in the hemoglobin α-chain, leading to diminished production of hemoglobin. A distinction is made between α- and β-thalassemia. Normally the α-globin chain is present as a double copy. Individuals with α-thalassemia has reduced or no synthesis of the alpha-chain. Alpha-thalassemia is usually detected in newborns by the presence of Hb-Bart's γ-chains. Beta-thalassemia is characterized by a reduction or absence of β-globin synthesis. Newborns with no β-globin chains will have no HbA and suffer from severe anemia. β0-thassemia or β-thalassemia major usually results in death during childhood. Individuals with reduced synthesis of β-globin chains will show reduced HbA, and in some case slightly elevated HbA2. As the prevalence of thalassemias is high, there is also a need to include thalassemia detection in routine neonatal screening.
There is thus an established need for inexpensive and easy-to-use screening assays, which can distinguish a healthy subject from a possibly afflicted subject needing further testing to diagnose a possible hemoglobinopathy.
There are, however, no clinically applicable methods that would give a simple afflicted/non-afflicted answer. Presently available methods for detecting hemoglobinopathies are all based on techniques that separate and identify all different variant hemoglobins present in the sample. Such methods, based on electrophoresis, isoelectric focusing (IEF) or HPLC are very labor-intensive and expensive and therefore not suitable for universal routine neonatal screening of hemoglobinopathies.
Electrophoretic methods, such as IEF are based on the fact that the variant Hbs have different electric charge. One commercially available product based on IEF is the Wallac RESOLVE® Neonatal Hemoglogin test kit, which is designed to separate cord blood hemoglogin on a thin layer gel to allow differentiation between sickle cell anemia and sickle cell trait. The separation of HbF from HbA permits differentiation of sickle cell anemia (HbSS) from sickle cell trait (HbAS). The preparation and separation of hemoglobin is accomplished through the application of a hemoglobin sample onto a precast agarose gel containing RESOLVE® Ampholytes, pH 6-8. RESOLVE® Ampholytes are composed of low molecular weight amphoteric molecules with varying isoelectric points. When an electrical current is applied to the gel, these molecules migrate through the gel to their isoelectric points (pl) along the gel, forming a stable pH gradient.
The hemoglobin variants also migrate through the gel until they reach the area where their individual pl:s equal the corresponding pH of the gel. At this point, the net charges on the variants are zero and migration ceases. The electric field counteracts diffusion and the hemoglobin variant forms a discrete thin band. Hemoglobin bands may be visualized using Perkin-Elmer's JB-2 STAINING SYSTEM®.
IEF is today probably the method of choice in most clinical laboratories for detecting hemoglobinopathies. However, this method requires the physical handling of numerous gels and is prone to pipetting errors. Moreover, the interpretation of the result is based on physical examination of the gels, and requires highly skilled technicians and experts interpreting the results. Furthermore, the testing laboratories are required to store the gels for a specified time period, requiring storage capacity and suitable facilities for safe storage.
A significant drawback to gel electrophoresis methods is their inability to identify Bart's hemoglobin. Thus α-thalassemias may be missed in this approach.
Another available method of diagnosing hemoglobinopathies is based on high performance liquid chromatography, HPLC, described e.g., in U.S. Pat. No. 4,108,603 and U.S. Pat. No. 5,719,053. These methods are not as labor-intensive as IEF but a considerable amount of work-load is still needed. The capacity of HPLC might also be limiting, if applied to routine neonatal screening programs, as simultaneous analysis of multiple samples is not possible.
A survey of available laboratory methods and international guidelines concerning the screening of hemoglobinopathies may be found in e.g., Clinical Chemistry, 46:8(B), 1284-1290, 2000 and in Guideline: The Laboratory Diagnosis of Haemoglobinopathies, British J Haematol, 101, 783-792,1998.
Absolute cost implications of the currently used hemoglobinopathy testing programs are difficult to estimate. Davies et al. in Health Technology Assessment 2000 estimate that the cost per baby when testing 50,000 newborns using IEF was £3.51 and £3.83 when using HPLC, that is £1.7 million for 500,000 births. At a prevalence of 1:2000 the costs thus rise to about £6700 per detected case. The overall cost per case detected has been approximated to £20,000 for the whole of United Kingdom.
Moscoso et al. describes in J Clin Lab Anal., 7(4), 214-19, 1993, an ELISA assay for differential diagnosis of hemoglobinopathies. They describe the use of monoclonal antibodies for normal and variant hemoglobins. Such a test is useful for diagnostic purposes and for specific identification of hemoglobin variants, but the need of including reagents for all known variants in the test, does not make it suitable for neonatal screening purposes.
Rosenthal et al, describes in Screening, 3(2), 67-76, 1994, monoclonal antibody assay HEMOCARD-kits for the identification of HbA, HbS, HbC and HbE. The test is useful for confirming the presence of variant hemoglobins, but again, it is not useful for neonatal screening purposes.
One common property of the above described methods for detecting hemoglobinopathies is the fact that they actually provide too much information to be useful as screening tests—in fact the current methods are useful as confirmatory diagnostic tests. An ideal screening program should identify only those at risk for a given disease, e.g. sickle cell disease (SCD). For example, in some health care systems sickle cell disease is the only hemoglobinopathy allowed to be reported. Thus, a screening test should primarily find the diseased group of interest. There is thus an established need of easy-to-use, inexpensive assays suitable for neonatal screening of hemoglobinopathies.